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WO2000070075A1 - Streptococcus pneumoniae yers - Google Patents

Streptococcus pneumoniae yers Download PDF

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Publication number
WO2000070075A1
WO2000070075A1 PCT/US2000/013320 US0013320W WO0070075A1 WO 2000070075 A1 WO2000070075 A1 WO 2000070075A1 US 0013320 W US0013320 W US 0013320W WO 0070075 A1 WO0070075 A1 WO 0070075A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
sequence
compnsmg
Prior art date
Application number
PCT/US2000/013320
Other languages
English (en)
Inventor
Magdalena Zalacain
Sanjoy Biswas
Martin K. R. Burnham
James R. Brown
Karen A. Ingraham
Alison F. Chalker
David J. Holmes
Richard L. Warren
Christopher M. Traini
Alexander P. Bryant
Andrea Marra
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation, Smithkline Beecham Plc filed Critical Smithkline Beecham Corporation
Publication of WO2000070075A1 publication Critical patent/WO2000070075A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their va ⁇ ants. agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the yerS (tRNA methyltransferases) family, as well as their va ⁇ ants. herein referred to as "yerS.” "yerS polynucleot ⁇ de(s),” and “yerS polypept ⁇ de(s)" as the case may be
  • Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact.
  • the present invention relates to yerS, in particular yerS polypeptides and yerS polynucleotides, recombinant mate ⁇ als and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of rmcrobial diseases, amongst others
  • the invention relates to methods for identifying agomsts and antagonists using the mate ⁇ als provided by the mvention, and for treating rmcrobial infections and conditions associated with such infections with the identified agonist or antagonist compounds
  • the invention relates to diagnostic assays for detecting diseases associated with rmcrobial infections and conditions associated with such infections, such as assays for detecting yerS expression or activity
  • the invention relates to yerS polypeptides and polynucleotides as desc ⁇ bed in greater detail below
  • the invention relates to polypeptides and polynucleotides of a yerS of Streptococcus pneumoniae, that is related by ammo acid sequence homology to B subtihs yerS polypeptide
  • the invention relates especially to yerS having a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • sequences recited in the Sequence Listmg below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ⁇ bopolynucleotides
  • a deposit comp ⁇ smg a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Indust ⁇ al and Marine Bacte ⁇ a Ltd (herem "NCIMB"), 23 St Machar D ⁇ ve, Aberdeen AB2 1RY, Scotland on 11 April 1996 and assigned deposit number 40794 The deposit was descnbed as Streptococcus pneumoniae 0100993 on deposit
  • Streptococcus pneumoniae 0100993 DNA library in E coll was similarly deposited with the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumoniae strain deposit is referred to herem as "the deposited strain” or as "the DNA of the deposited strain"
  • the deposited strain comp ⁇ ses a full length yerS gene
  • the sequence of the polynucleotides comp ⁇ sed m the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any desc ⁇ ption of sequences herem
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merely as convemence to those of skill m the art and is not an admission that a deposit is required for enablement. such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is comp ⁇ sed m the deposited strain
  • yerS polynucleotide sequences in the deposited strain such as DNA and RNN and ammo acid sequences encoded thereby
  • YerS polypeptide of the mvention is substantially phylogenetically related to other proteins of the yerS (tR ⁇ A methyltransferases) family
  • yerS polypeptides of Streptococcus pneumoniae referred to herem as "yerS” and “yerS polypeptides” as well as biologically, diagnostically, prophylactically, clirucally or therapeutically useful va ⁇ ants thereof, and compositions comp ⁇ smg the same
  • the present mvention further provides for an isolated polypeptide that (a) comp ⁇ ses or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide compnsmg or consistmg of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (c) a polypeptide encoded by an isolated polynucleotide comp ⁇ smg or consistmg of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ED NO 2, over the entire
  • X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, and at the carboxyl terminus
  • Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides
  • Ri and R3 are any ammo acid residue or modified ammo acid residue
  • m is an mteger between 1 and 1000 or zero
  • n is an mteger between 1 and 1000 or zero
  • R 2 is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R 2 is o ⁇ e ted so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the nght, covalently bound to R3 Any stretch of ammo acid residues denoted by
  • a polypeptide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Prefened fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Streptococcus pneumoniae are also prefened. Further prefened are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Further prefened fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of
  • SEQ ID NO:2 or an isolated polypeptide comprising an amino acid sequence having at least 15,
  • Fragments of the polypeptides of the invention may be employed for producing the conesponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • the polynucleotide comprises a region encoding yerS polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] that includes a full length gene, or a variant thereof.
  • SEQ ID NO:l a sequence set out in Table 1 [SEQ ID NO:l] that includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Streptococcus pneumoniae .
  • isolated nucleic acid molecules encoding and/or expressmg yerS polypeptides and polynucleotides, particularly Streptococcus pneumoniae yerS polypeptides and polynucleotides. including, for example, unprocessed RNAs, ⁇ bozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • RNAs unprocessed RNAs, ⁇ bozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions comp ⁇ smg the same
  • Another aspect of the mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a yerS polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • a yerS polypeptide from
  • Streptococcus pneumoniae comprising or consistmg of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof
  • a polynucleotide of the mvention encoding yerS polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a using Streptococcus pneumoniae 0100993 cells as starting mate ⁇ al, followed by obtaining a full length clone
  • a polynucleotide sequence of the mvention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]
  • typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 m E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, denved from a partial sequence
  • each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art
  • the polynucleotide of SEQ ID NO 1 between nucleotide number 1 and the stop codon that begins at nucleotide number 1630 of SEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2
  • the present mvention provides for an isolated polynucleotide compnsmg or consistmg of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97% still more preferably at least 98%, yet still more preferably at least 99%, even still more preferably at least 99 5% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire lenght of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encodmg a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encoding a polypeptide of the present mvention may be obtained by a process that compnses the steps of screening an approp ⁇ ate library under stringent hybndization conditions with a labeled or detectable probe consistmg of or comp ⁇ smg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones comp ⁇ smg said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open reading frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide of the mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNN introns, and polyadenylation signals
  • polynucleotides of the mvention also mclude, but are not limited to, polynucleotides comp ⁇ smg a structural gene and its naturally associated sequences that control gene expression
  • a prefened embodiment of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1630 set forth m SEQ ID NO 1 of Table 1, both of that encode a yerS polypeptide
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
  • X-(R ⁇ ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occunence of Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof.
  • R is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either Ri and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • m and/or n is an integer between 1 and 1000.
  • Other prefened embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polynucleotide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae yerS having an amino acid sequence set out in Table 1 [SEQ ID NO:2].
  • polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may comprise coding and/or non-coding sequences .
  • the invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention. Further particularly prefened embodiments are polynucleotides encoding yerS variants, that have the amino acid sequence of yerS polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
  • Prefened isolated polynucleotide embodiments also include polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO: l.
  • polynucleotide fragments such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding yerS polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides.
  • Most highly prefened are polynucleotides that comprise a region that is at least 95 % are especially prefened.
  • those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% being the more prefened.
  • Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ED NO: 1] .
  • polynucleotides that hybridize, particularly under stringent conditions, to yerS polynucleotide sequences, such as those polynucleotides in Table 1.
  • the invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein.
  • the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein.
  • stringent conditions and “stringent hybridization conditions” mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences.
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C.
  • the mvention also provides a polynucleotide consistmg of or compnsmg a polynucleotide sequence obtained by screening an appropnate library compnsmg a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybndization conditions with a probe havmg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide mclude, for example, probes and primers fully descnbed elsewhere herem
  • the polynucleotides of the mvention may be used as a hybndization probe for RNN cD ⁇ A and genomic D ⁇ A to isolate full-length cD ⁇ As and genomic clones encodmg yerS and to isolate cD ⁇ A and genomic clones of other genes that have a high identity, particularly high sequence identity, to a yerS gene
  • Such probes generally will compnse at least 15 nucleotide residues or base pairs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularly prefened probes will have at least 20 nucleotide residues or base pa rs and will have lee than 30 nucleotide residues or base pairs
  • a coding region of a yerS gene may be isolated by screening usmg a D ⁇ A sequence provided m Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe
  • a labeled ohgonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNN genomic DNA or mRNA to determine which members of the library the probe hybndizes to
  • polynucleotides of the mvention that are ohgonucleotides denved from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herem as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or m part are transcnbed m bactena m infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional ammo or carboxyl-terminal ammo acids, or ammo acids intenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case in v vo, the additional ammo acids may be processed away from a mature protein by cellular enzymes
  • a precursor protein, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques .
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of streptococci, staphylococci, enterococci E. coli, streptomyces, cyanobacteria, Bacillus subtihs, and Streptococcus pneumoniae
  • fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus
  • insect cells such as cells of Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gymnosperm or angiosperm.
  • vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
  • viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
  • the expression system constructs may compnse control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techmques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification Well known techmques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
  • This mvention is also related to the use of yerS polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of yerS polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the yerS gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techmques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily mate ⁇ als
  • Polynucleotides from any of these sources, particularly DNA or RNN may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique p ⁇ or to analysis
  • R N particularly mR ⁇ N cD ⁇ A and genomic D ⁇ A may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species Pomt mutations can
  • an anay of ohgonucleotides probes comp ⁇ smg yerS nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification
  • Anay technology methods are well known and have general applicability and can be used to address a vanety of questions in molecular genetics including gene expression, genetic linkage, and genetic vanabihty (see, for example, Chee et al , Science, 274 610 (1996))
  • the present mvention relates to a diagnostic kit that compnses (a) a polynucleotide of the present mvention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b), (c) or (d) may compnse a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1 , that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide
  • Organisms, particularly infectious organisms, carrymg mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem
  • a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessmg a different, second different phenotype can also be determined If a mutation is observed in some or all organisms possessmg the first phenotype but not m anv orga sms possessmg the second phenotype, then the mutation .s likely to be the causative agent of the first phenotype
  • a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to allow for serotyping, for example
  • RT-PCR can be used to detect mutations in the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNN cD ⁇ A or genomic D ⁇ A may also be used for the same purpose, PCR
  • PCR p ⁇ mers complementary to a polynucleotide encoding yerS polypeptide can be used to identity and analyze mutations
  • the mvention further provides these p ⁇ mers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These p ⁇ mers may be used for, among other thmgs, amplifying yerS
  • the p ⁇ mers ma ⁇ be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide ma ⁇ then be subject to va ⁇ ous techmques for elucidation of the polynucleotide sequence
  • mutations m the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Streptococcus pneumoniae, compnsmg determining from a sample denved from an individual, such as a bodily matenal, an mcreased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a yerS polynucleotide can be measured usmg any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods
  • a diagnostic assay m accordance with the mvention for detecting over-expression of yerS polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a yerS polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art
  • Assay techmques that can be used to determine levels of a yerS polypeptide, m a sample denved from a host, such as a bodily matenal
  • Such assay methods mclude radioimmunoassays, competitive-bmdmg assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and gands in, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures
  • substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Co gan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991)
  • Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, including many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g ,inhibit) the function of the polypeptide or polynucleotide Accordingly, m a further aspect, the present mvention provides for a method of
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem of the polypeptide by means of a label directly or mdirectly associated with the candidate compound Alternatively, the screening method may mvolve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropnate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed m screening methods for mverse agomsts, in the absence of
  • polypeptides and antibodies that bind to and/or mteract with a polypeptide of the present mvention may also be used to configure screemng methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the invention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of yerS polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal.
  • the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising yerS polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a yerS agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the yerS polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i.e., without inducing the effects of yerS polypeptide are most likely to be good antagonists.
  • Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system.
  • Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in yerS polynucleotide or polypeptide activity, and binding assays known in the art.
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
  • a radioactive isotope for instance, ⁇ I
  • chemically modified for
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational conelation time or tumbling rate.
  • Protein complexes such as formed by yerS polypeptide associating with another yerS polypeptide or other polypeptide, labeled to comprise a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monome ⁇ c protem It is preferred that this method be used to charactenze small molecules that disrupt polypeptide complexes
  • Fluorescence energy transfer may also be used charactenze small molecules that interfere with the formation of yerS polypeptide drmers, tnmers, tetramers or higher order structures, or structures formed by yerS polypeptide bound to another polypeptide YerS polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block drmenzation will inhibit fluorescence energy transfer
  • YerS polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protem can then passed over the yerS polypeptide -coated surface and specific bmdmg can be detected in real-time by monitoring the change m resonance angle caused by a change m local refractive mdex
  • This technique can be used to charactenze the effect of small molecules on kinetic rates and equihbnum bmdmg constants for yerS polypeptide self-association as well as an association of yerS polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to charactenze the mteraction between an association of yerS polypeptide with another yerS polypeptide or a different polypeptide YerS polypeptide
  • identifying compounds that bmd to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the binding or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of
  • an assay for yerS agonists is a competitive assay that combmes yerS and a potential agomst with yerS-binding molecules, recombinant yerS bmdmg molecules, natural substrates or hgands. or substrate or ligand mimetics.
  • YerS can be labeled, such as by radioactivity or a colo ⁇ metnc compound, such that the number of yerS molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagomst
  • a polypeptide and/or polynucleotide of the present mvention may also be used m a method for the structure-based design of an agomst or antagomst of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducmg the three-dimensional structure for the likely reactive s ⁇ te(s), bmdmg s ⁇ te(s) or mot ⁇ f(s) of an agomst or antagomst,
  • the present mvention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of yerS polypeptide and/or polynucleotide
  • expression of the gene encodmg endogenous yerS polypeptide can be inhibited usmg expression blocking techmques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted agamst transcnption and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 ⁇ n Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides that form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney t ⁇ / , Science (1988) 241 456, Dervan et o/ , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the
  • Each of the polynucleotide sequences provided herem may be used m the discovery and development of antibactenal compounds
  • the encoded protem upon expression, can be used as a target for the screenmg of antibactenal drugs
  • the polynucleotide sequences encodmg the ammo terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the codmg sequence of interest
  • the mvention also provides the use of the polypeptide, polynucleotide, agomst or antagomst of the mvention to mterfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the mvention may be used m the prevention of adhesion of bactena, m particular gram positive and/or gram negative bactena, to eukaryotic, preferably mammalian, extracellular matnx protems on in-dwelling devices or to extracellular matnx protems m wounds, to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matnx protems and bactenal yerS protems that mediate tissue damage and/or, to block the normal progression of pathogenesis m infections initiated other than by the implantation of in-dwelling devices or by other surgical
  • yerS agomsts and antagomsts preferably bactenstatic or bactencidal agomsts and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • Hehcobacter pylori herem "H pylori" bactena infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm)
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastnc adenocarcinoma, classifying the bactenum as a Group I (definite) carcinogen
  • Prefened antimicrobial compounds of the mvention found usmg screens provided by the mvention, or known in the art, particularly nanow-spectrum antibiotics, should be useful
  • Bodily matenal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, mcludmg , for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid
  • “Host cell(s)” is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Gnffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis in Molecular Biology, von Hemje, G
  • Parameters for polypeptide sequence companson mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff. Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps)
  • Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97, 98, 99. 99 5 or 100% identity to the reference sequence of SEQ ID NO 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either mdividually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides m SEQ ID NO 1
  • y is 0 95 for 95%. 0 97 for 97%, 0 98 for 98%, 0 99 for 99%, 0 995 for 99 5% or 1 00 for 100%.
  • any non-integer product of x n and y is rounded down to the nearest mteger pnor to subtracting it from x n
  • Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may create nonsense, rrussense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either mdividually among the ammo acids in the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids m SEQ ID NO 2 by the
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids m SEQ ID NO 2.
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-integer product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • “Ind ⁇ v ⁇ dual(s)" means a multicellular eukaryote, mcludmg, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a pnmate. and a human
  • Isolated means altered “by the hand of man” from its natural state, i e , if it occurs m nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present m a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is mtroduced mto an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may be living or non-living
  • Organicgan ⁇ sm(s) means a (I) prokaryote, mcludmg but not limited to, a member of the genus
  • Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobacterium, Neissena, Haemophilus, Actinomycetes Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella Acinetobacter, Erys ⁇ elothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostndium, Treponema, Eschench a, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borreha and Mycoplasma, and further mcludmg, but not limited
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxy ⁇ bonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotide(s)” mclude, without limitation, single- and double-stranded DNN D ⁇ A that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, single- and double-stranded R ⁇ N and R ⁇ A that is mixture of single- and double-stranded regions, hyb ⁇ d molecules compnsmg D ⁇ A and R ⁇ A that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide as used herem refers to tnple-stranded regions compnsmg R ⁇ A or
  • Polypeptide(s) refers to any peptide or protem comp ⁇ smg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds
  • Polypeptide(s) refers to both short chains, commonly refened to as peptides, ohgopeptides and ohgomers and to longer chains generally refened to as proteins
  • Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids
  • Polypeptide(s)” mclude those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of
  • covalent attachment of a hpid or hpid de ⁇ vative covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation.
  • Polypeptides may be branched or cychc, with or without branching Cychc, branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention 'Na ⁇ ant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed below
  • va ⁇ ants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combmation
  • a vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae m E coli
  • the sequencmg data from two or more clones compnsmg overlappmg Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routme methods, for example Methods 1 and 2 below Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods Method 1
  • Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate accordmg to standard procedures
  • DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library
  • the library is amplified by standard procedures
  • Method 2 Total cellular DNA is partially hydrolyzed with a one or a combmation of restnction enzymes appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, A , Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library The library is amplified by standard procedures Example 2 yerS Characterization
  • the S. pneumoniae yerS gene is expressed during infection in a respiratory tract infection model
  • RNAase free, DNAase free, DNA and protem free preparations of RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) usmg unique pnmer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993 a) Isolation of tissue infected with Streptococcus pneumomae 0100993 from
  • Streptococcus pneumomae 0100993 is seeded onto TSA (Tryptic Soy Agar, BBL) plates containmg 5% horse blood and allowed to grow overnight at 37°C m a C02 mcubator Bactenal growth is scraped mto 5 ml of phosphate-buffered saline (PBS) and adjusted to an A600 ⁇ 0 6 (4 x 106/ml) Mice (male CBA/J-1 mice, approximately 20g) were anaesthetized with isoflurane and 50 microhters of the prepared bactenal moculum is delivered by mtranasal mstillation Animals are allowed to recover and observed twice daily for signs of monbundancy Forty-eight hours after infection the animals are euthamzed by carbon dioxide overdose and their torsos swabbed with ethanol and then RNAZap The torso is then opened, and the lungs are aseptically removed Half of each pair
  • RNA isolation is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA yields
  • 2 2M formaldehyde gels are run and vacuum blotted to Hybond-N (Amersham)
  • the blot is then hybndised with a 32P-labelled oligonucletide probe, of sequence 5' AACTGAGACTGGCTTTAAGAGATTA 3' [SEQ ID NO 3], specific to 16S rRNA of Streptococcus pneumomae
  • the size of the hybndismg band is compared to that of control RNA isolated from m vitro grown Streptococcus pneumomae 0100993 m the Northern blot Conect sized bactenal 16S rRNA bands can be detected m total RNA samples which show degradation of the mammalian
  • DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 umts of RNAase-free DNAasel (GenHunter) m the buffer supplied m a final volume of 57 microhters
  • the DNAase was mactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) accordmg to the manufacturers protocol
  • DNAase treated RNA was resuspended m 100 microhtres of DEPC treated water with the addition of Rnasin as descnbed before d)
  • PCR reactions are set up on ice m 0 2ml tubes by addmg the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR pnmers (optimally 18- 25 basepairs m length and designed to possess similar annealmg temperatures), each pnmer at lOmM initial concentration, and 5 microhtres cDNA
  • PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 oC, then 50 cycles of 30 seconds each at 94 oC, 50 oC and 72 oC followed by 7 minutes at 72 oC and then a hold temperature of 20 oC (the number of cycles is optimally 30-50 to determme the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made), 10 microhtre aliquots are then run out on 1% 1 x TBE gels stained with ethidmm bromide, with PCR product, if present, sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternatively if the PCR products are convemently labelled by the use of a labelled PCR pnmer (e g labelled at the 5'end with a dye) a suitable aliquot of the PCR product is run out on
  • RT/PCR controls may mclude +/- reverse transcnptase reactions, 16S rRNA pnmers or DNA specific pnmer pairs designed to produce PCR products from non-transcnbed Streptococcus pneumomae 0100993 genomic sequences
  • PCR failures and as such are unrnformative Of those which give the conect size product with DNA PCR two classes are distinguished m RT/PCR 1 Genes which are not transcnbed m vivo reproducibly fail to give a product m RT/PCR, and 2 Genes which are transcnbed m vivo reproducibly give the conect size product m RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) m -RT controls
  • m RT/PCR 1 Genes which are not transcnbed m vivo reproducibly fail to give a product m RT/PCR
  • the yerS gene when mutated causes attenuation in a S.pneumoniae respiratory tract infection model.
  • a S pneumoniae yerS mutant was generated as descnbed below When tested in a respiratory tract infection as descnbed below, the mutant was found to be 4 logs attenuated compared to an infection with the wild-type parent strain a) Procedure for generating S pneumomae allehc replacement mutants
  • a DNA construct is generated by PCR, consistmg of 500bp chromosomal DNA fragments flanking an erythromycin resistance gene
  • the chromosomal DNA sequences are usually the 500bp preceding and following the gene of mterest
  • allehc replacement cassette is introduced mto S pneumomae R6 or S pneumomae 100993 by transformation Competent cells are prepared accordmg to published protocols
  • DNA is mtroduced mto the cells by incubation of 500ng of allehc replacement cassette with 10 6 cells at 30oC for 30 minutes
  • the cells are transfened to 37oC for 90 minutes to allow expression of the erythromycin resistance gene
  • Cells are plated in agar containmg lug erythromycm per ml Following mcubation at 37oC for 36 hours, any observed colonies are picked and grown overnight in Todd-Hewitt broth supplemented with 0 5% yeast extract
  • chromosomal DNA is prepared from these cells and examined usmg diagnostic PCR Ohgonucleotides designed to hybndize to sequences within the allehc replacement cassette are used m conjunction with DNA pnmers hybndizmg to chromosomal sequences outside the cassette to generate DNA products amplified by PCR of charactenstic size This chromosomal DNA is also subject to Southern analysis m order to venfy that the appropnate chromosomal DNA reanangement has occurred
  • the defective strain is grown for many generations m the absence of selective pressure and then assayed for its ability to grow in the absence and presence of erythromycm b) Procedures for respiratory tract infection with Streptococcus pneumomae
  • Bacteria for infection are prepared by inoculation of tryptic soy agar plates containmg 5% sheep blood from frozen stocks and overnight growth at 37°C m 5% C02 Bacteria are recovered from the plates, resuspended m phosphate-buffered salme (PBS) and adjusted to A600-0 8 - 1 0 (approximately 107 - 108 cfu/ml) Animals (male CBA/J mice, 14 - 16g) are anaesthetized with isoflurane (3%), and 50 microhters of the prepared bactenal moculum is administered by mtranasal instillation usmg a pipetman No pam is inflicted on the animals at any point during this procedure, and no other anaesthetic is used To simulate the state m immunocompromised patients the immune system of the animals may be suppressed by agents such as cyclophosphamide (150mg/kg l p on day -4 followed by l

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Abstract

L'invention porte sur des polypeptides yerS et sur des polynucléotides les codant, ainsi que sur des procédés de production de ces polypeptides par des techniques de recombinaison. L'invention porte également sur des procédé d'utilisation de ces polypeptides yerS permettant de cribler des composés antibactériens.
PCT/US2000/013320 1999-05-17 2000-05-12 Streptococcus pneumoniae yers WO2000070075A1 (fr)

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US31352699A 1999-05-17 1999-05-17
US09/313,526 1999-05-17

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WO2000070075A1 true WO2000070075A1 (fr) 2000-11-23

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PCT/US2000/013320 WO2000070075A1 (fr) 1999-05-17 2000-05-12 Streptococcus pneumoniae yers

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018931A2 (fr) * 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Polynucleotides et sequences de streptococcus pneumoniae

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018931A2 (fr) * 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Polynucleotides et sequences de streptococcus pneumoniae

Non-Patent Citations (1)

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Title
VERMA ET AL.: "Gene therapy - promises, problems and prospects", NATURE,, vol. 389, 18 September 1997 (1997-09-18), pages 239 - 242, XP002930100 *

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